PROJECT SUMMARY Protein-protein and protein-nucleic acid interactions have been regarded as ?undruggable? despite the pivotal roles these biomolecule complexes play in cell signaling and disease development. The overall objective of this current proposal is to establish a general strategy to develop exogenous chemical probes with high specificity and potency to inhibit protein-protein, protein-RNA, and protein-DNA interactions involving membrane proteins. We will utilize the toll-like receptors (TLRs) protein family as a model system for studying these protein-protein and protein-nucleic acid interactions because of their essential roles in mediating the inflammatory response and in the development of various diseases. Further, homologous, TLR-family proteins can be activated through the formation of a variety of protein- protein and protein-nucleic acid complexes, providing an excellent platform for testing selectivity and specificity. With systematic high-throughput screening and structure-based design expertise established in our lab, we are well positioned to develop novel exogenous chemical probes for these challenging nonetheless important membrane protein complexes. We will work with Drs. Ian Wilson (TLR3/dsRNA and TLR5/Flagellin), Toshiyuki Shimizu (TLR7/ssRNA, TLR8/ssRNA, and TLR9/ssDNA), and Jie-Oh Lee (TLR4/MD-2 and TLR1/TLR2). These investigators count among those who first successfully solved the high-resolution structures of various TLR protein-protein and protein-nucleic acid complexes. Our collaborators will provide assistance in biophysical characterizations and structural analyses of the identified TLR ligands. We will also work closely with Drs. Linda Watkins and Sebastian Zimmer, who have extensive expertise in animal modeling, for testing of the identified inhibitors. These proposed studies, if successful, are projected to yield significant novel outcomes. First, the results will further the understanding of the mechanism of clinically relevant TLR-mediated inflammation. Second, successful development of highly specific and potent small molecule inhibitors/activators will validate protein-protein, protein-RNA, and protein-DNA complexes as feasible targets for drug discovery. Finally, our cross- disciplinary approaches would lay the groundwork for the development of next-generation probing technologies for challenging membrane protein targets. We expect much synergy from the combination of different chemical probe development themes within our lab through the MIRA mechanism. Importantly, potential MIRA funding will significantly increase our flexibility to take on new, ambitious directions in the fast-evolving research field of immunomodulator development and membrane protein chemical biology. A potential MIRA award will also allow me to increase overall scientific productivity and educational commitments to mentor junior scientists in our productive research group. !